A conventional capillary array electrophoresis system is configured to perform a high-throughput analysis on biological samples, e.g., DNA sequencing, using a highly sensitive laser-induced fluorescence detection method. In particular, the samples to be analyzed either possess fluorescing functional groups (fluorophores) in their molecular structure or are tagged with fluorescent dyes. These samples are then excited with a laser beam which causes the samples to emit fluorescence light. The emitted fluorescence light is detected and subsequently analyzed.
The samples are illuminated by the laser beam either while they are still migrating through the capillaries, i.e., on-column detection, or after they elute from output ends of the capillaries, i.e., sheath-flow detection, as described by Dovichi et al. (U.S. Pat. No. 5,741,412).
For the on-column detection method, samples in the confocal microscope scanning method can be used as described in Mathies el al. (U.S. Pat. No. 5,274,240) In this method, samples in each capillary are sequentially excited and detected by a confocal scanning system. In another method, as described by Yeung et al,(U.S. Pat. No. 5,741,411) all the capillaries are illuminated by a laser beam and monitored by a 2-dimensional charged couple device (CCD) simultaneously.
FIG. 1 illustrates a conventional on-column detection system 1 that includes a laser light source 3 illuminating a capillary array 5 and samples therein and a camera lens 7 receiving the emitted light from the samples. Subsequently, the received light from the samples is captured by a CCD and analyzed.
FIG. 2 shows an intensity profile, amounts of light received across a viewing field of the camera 7. More specifically, the measurements are made by illuminating a laser beam on the array of capillaries 5 having the same quantity of samples migrating through each of the capillaries. The view field of the camera 7 is about 2 cm, i.e., the width of a 96 capillary array comprising capillaries with 200 μm outside diameters (o.d.)laid side by side. The position of 300 in FIG. 2 corresponds to the center of the array.
The resulting intensity profile shows that the amount of the light received from the location near the center of the array is more than that from capillaries at the periphery of the array.
At least two aspects of the conventional system 1 cause this effect. First, the laser beam has a Gaussian beam profile. In other words, a laser beam produced by a conventional laser illuminates the capillaries in the middle portion with about 1.5 times more intensity than the capillaries at the periphery of the array. Second, the amount of light captured by the camera varies based on the location of the capillaries. In particular, the amount of light received by the camera from a unit area of the capillaries at the periphery of the array is less than that from a unit area of the capillary at the center of the array, when an identical amount of light is emitted by the samples within the capillaries in each of the unit areas.
The above discussed shortcomings of the conventional system produce a non-uniform intensity profile. For instance, the amounts of light received from the center capillaries and periphery capillaries can differ by a factor of 2-4, as shown in FIG. 2.
The non-uniform intensity profile is not desirable, because in order to obtain sufficient amounts of light from the capillaries at the periphery of the array, the strength of the laser beam illuminating the center of the array may saturate the camera. Further, in order to process and analyze the data collected under this condition for capillary-to-capillary comparison and quantification, the subsequent analysis process becomes complicated.